In the present study, there are three major findings. First, under physiological conditions, beta-arrestin1 and beta-arrestin2 physically interact with each other. Second, phosphorylation of beta-arrestin1 plays a crucial role in the formation of the beta-arrestin complex and subsequent recruitment of PP2A. Third, ablation of beta-arrestin2 leads to markedly increased phosphorylation of beta-arrestin1 which promotes cell survival and cell growth via elevated activation of ERK1/2. (1) Co-localization and co-immunoprecipitation of beta-arrestin1 and beta-arrestin2 To investigate the potential relationship between beta-arrestin1 and beta-arrestin2, we expressed both beta-arrestin family members at a matched level in cultured mouse embryonic fibroblasts (MEFs) derived from beta-arrestin1 and beta-arrestin2 double knockout mice. Using confocal immunocytochemical imaging, we visualized a similar intracellular distribution pattern of specific immunofluorescent signals of beta-arrestin1 and beta-arrestin2 and an excellent pixel-to-pixel correction when the images were emerged in cultured mouse MEFs, suggesting these beta-arrestins are co-localized. To directly demonstrate physical interaction of beta-arrestin1 with beta-arrestin2, we performed immunoprecipitation and Western blotting. Total cellular proteins containing both beta-arrestin subtypes were first immunoprecipitated with either a beta-arrestin1 or beta-arrestin2 polyclonal antibody. The pull-down of beta-arrestin1 by anti-beta-arrestin2 in the immunoprecipitation was then confirmed by Western blot. (2) Ablation of beta-arrestin2 increases the phosphorylation level of beta-arrestin1 and reduces beta-arrestin1/ PP2A complex formation To explore the functional consequence of the intermolecular interaction of beta-arrestin1 and beta-arrestin2, we examined the potential impact of beta-arrestin2 ablation on beta-arrestin1 phosphorylation in MEFs derived from beta-arrestin2 knockout (KO) mice. To our surprise, the lack of beta-arrestin2 led to a profound increase in beta-arrestin1 phosphorylation level. Adenoviral gene transfer of beta-arrestin2 fully normalized -arrestin1 phosphorylation status, indicating that the hyper-phosphorylation of beta-arrestin1 is specifically attributable to the absence of beta-arrestin2 rather than an adaptive response due to beta-arrestin2 null background. Next, we found that endogenous beta-arrestin1 and PP2A form a complex in MEFs derived from wild type (WT) mice, as evidenced by their abundant co-immunoprecipitation. The association of beta-arrestin1 with PP2A was strikingly impaired in beta-arrestin2 deficient MEFs. Adenoviral gene transfer of beta-arrestin2 rescued the interaction of beta-arrestin1 with PP2A, indicating that beta-arrestin2 is required for the formation of PP2A/beta-arrestin1 complex. These data suggest that beta-arrestin2-dependent interaction of PP2A with beta-arrestin1 may sever as an important mechanism responsible for beta-arrestin1 dephosphorylation and the termination of beta-arrestin1-mediated signaling. We further determined whether altered beta-arrestin1 phosphorylation status affects its interaction with beta-arrestin2 and PP2A. A dominant negative beta-arrestin1 mutant (S412A) markedly impaired beta-arrestin heterodimerization and reduced the recruitment of PP2A by beta-arrestin1. These data corroborate the importance of beta-arrestin1 phosphorylation in its intermolecular interaction with beta-arrestin2 that subsequently recruit PP2A to the beta-arrestin complex. (3) Deficiency of beta-arrestin2 increases cell viability, rendering cells resistant to oxidative stress-induced cell death. The next key question is what is the biological significance of the negative regulation of beta-arrestin1 phosphorylation by beta-arrestin2? To address this question, we characterized cell survival and cell growth in WT MEFs or those lacking either beta-arrestin1 or 2. Interestingly, deficiency of beta-arrestin2, but not beta-arrestin1, rendered cells resistant to H2O2-induced cell death. H2O2-induced reduction in cell viability was fully restored in beta-arrestin2 KO MEFs infected by an adenoviral expression of beta-arrestin2, indicating that the resistance of beta-arrestin2 KO cells to oxidative stress is a direct consequence of the gene ablation instead of an adaptive response. In addition, deficiency of beta-arrestin2 but not beta-arrestin1 markedly enhanced MEF growth rate, suggesting the coexisted beta-arrestin1 and beta-arrestin2 elicit different functional roles. (4) Beta-arrestin1 phosphorylation positively correlates with activation of ERK1/2. To define the mechanism underlying the prosurvival and pro-growth effects of beta-arrestin2 ablation, we examined major signaling pathways involved in the regulation of cell fate, in particular, ERK1/2 MAPK pathway. ERK1/2 phosphorylation level was overtly augmented in beta-arrestin2 deficient cells, and restored to normal level when beta-arrestin2 was reintroduced. Inhibition of protein phosotases with okadaic acid (OA) led to a dose-dependent increase in beta-arrestin1 phosphorylation which was accompanied by a proportional increase in EKR1/2 phosphorylation (activation). In contrast, expression of the dominant negative beta-arrestin1 (S412A) mutant suppressed ERK1/2 activation. These results indicate that beta-arrestin2 ablation-associated increase in ERK1/2 activation is a consequence of hyper-phosphorylation of beta-arrestin1. Importantly, inhibition ERK1/2 activity with PD98059 (10 mM) blocked beta-arrestin2 ablation-induced cell resistance to oxidative stress, indicating that the pro-survival effect of beta-arrestin2 ablation is mediated by enhanced phosphorylation of beta-arrestin1 and subsequent activation of ERK1/2. These findings not only define beta-arrestin2 as a powerful endogenous negative regulator of beta-arrestin1 phosphorylation and biological function, but also highlight the importance of the balance between the coexisted beta-arrestin1 and beta-arrestin2 in maintaining normal cell growth and cell viability.